Allosteric control of nuclear hormone receptors

ABSTRACT

Heterodimerization is a common paradigm among eucaryotic transcription factors, though it remains unclear how individual monomers contribute to the overall transcriptional activities of the complex. The 9-cis retinoic acid receptor (RXR) serves as a common heterodimerization partner for several nuclear receptors including the thyroid hormone (T 3 R), retinoic acid (RAR) and vitamin D receptors. A strategy has been devised to examine the transcriptional properties of each receptor individually or when tethered to a heterodimeric partner. It has been found that the intrinsic activity of RXR is masked in RXR-T 3 R and RXR-RAR heterodimers. In contrast, a novel RXR-Nurrl heterodimer described herein is highly responsive to RXR ligands, suggesting that different partners exert unique allosteric control over the RXR response. These findings establish a novel 9-cis retinoic acid response pathway and resolve the paradox as to how T 3 R, RAR and VDR contribute to distinct physiologic pathways while sharing a common RXR subunit.

FIELD OF THE INVENTION

[0001] The present invention relates to intracellular receptors, andmethods for the modulation thereof. In a particular aspect, the presentinvention relates to novel heterodimeric complexes. In another aspect,the present invention relates to methods for modulating processesmediated by retinoid X receptor and/or orphan receptor Nurrl.

BACKGROUND OF THE INVENTION

[0002] Heterodimerization is a common theme in eucaryotic regulatorybiology. Indeed, a number of transcription factor families have beendefined by their characteristic dimerization interface. These includethe leucine zipper (e.g. fos, jun, CREB, C/EBP; see, for example, Lamband McKnight, in Trends Biochem. Sci. 16:417-422 (1991)),helix-loop-helix (e.g. myc, max, MyoD, E12, E47; see, for example, Amatiand Land, in Curr. Opin. Genet. Dev. 4:102-108 (1994)), rel (NFκB,dorsal; see, for example, Blank et al., in Trends Biochem. Sci.17:135-140 (1992)), ankyrin (GABP; see, for example, Brown and McKnight,in Genes Dev. 6:2502-2512 (1992)), and the nuclear receptorsuperfamilies (see, for example, Evans, in Science 240:889-895 (1988),and Forman and Samuels, Mol. Endocrinol. 4:1293-1301 (1990)). Detailedanalyses of these proteins have shown that heterodimerization producesnovel complexes that bind DNA with higher affinity or alteredspecificity relative to the individual members of the heterodimer (see,for example, Glass, in Endocr. Rev. 15:391-407 (1994)). Indeed, littleis known about the contributions of each monomer toward thetranscriptional properties of the complex.

[0003] Nuclear hormone receptors are characterized by a central DNAbinding domain (DBD, see FIG. 1), which targets the receptor to specificDNA sequences, known as hormone response elements (HREs) . The retinoicacid receptor (RAR), the thyroid hormone receptor (T₃R) , the vitamin D₃receptor (VDR) and the fatty acid/peroxisome proliferator activatedreceptor (PPAR) preferentially bind to DNA as heterodimers with a commonpartner, the retinoid X (or 9-cis retinoic acid) receptor (RXR; see, forexample, Yu et al., in Cell 67:1251-1266 (1991); Bugge et al., in EMBOJ. 11:1409-18 (1992); Kliewer et al., in Nature 355:446-449 (1992); Leidet al, in Cell 68:377-395 (1992); Marks et al., in EMBO J. 11:1419-1435(1992); Zhang et al., in Nature 355:441-446 (1992); and Issemann et al.,in Biochimie. 75:251-256 (1993).

[0004] Naturally occurring HREs are composed of direct repeats (i.e.,DRs; see Umesono et al., in Cell 65:1255-1266 (1991), inverted repeats(i.e., IRs; see Umesono et al., in Nature 336:262-265 (1988), andWilliams et al. in J. Biol. Chem. 266:19636-19644 (1991)), and/oreverted repeats (ERs; see Baniahmad et al., in Cell 61:505-514 (1990);Farsetti et al., in J. Biol. Chem. 267:15784-15788 (1992); Raisher etal., in J. Biol. Chem. 267:20264-20269 (1992); or Tini et al., in GenesDev. 7:295-307 (1993)) of a degenerate X_(n)-AGGTCA core-site.

[0005] The DNA binding domain (DBD) contains two helical regions, one ofwhich serves as a recognition helix that makes base-specific contactswithin the major groove of the core-site (see, for example, Luisi etal., in Nature 352:497-505 (1991) and Schwabe et al., in Cell 75:567-578(1993)). A third helix has been identified in some receptors which makesadditional minor groove contacts in the 5′ portion of the core-bindingsite, X_(n) (see, for example, Wilson et al., in Science 256:107-110(1992) or Lee et al., in Science 260:1117-1121 (1993)).

[0006] In direct repeats (DR, head-to-tail arrangement) the X_(n)sequence also serves as a gap which separates the two core-bindingsites. Spacers of 1, 3, 4 and 5 nucleotides serve as preferred responseelements for heterodimers of RXR with PPAR, VDR, T₃R and RAR,respectively (see, for example, Naar et al., in Cell 65:1267-1279(1991); Umesono et al., 1991, supra; Kliewer et al., in Nature358:771-774 (1992); and Issemann et al., supra). The optimal gap lengthfor each heterodimer is determined by protein-protein contacts whichappropriately position the DBDs of RXR and its partner (see, forexample, Kurokawa et al., in Genes Dev. 7:1423-1435 (1993); Perlmann etal., in Genes Dev. 7:1411-1422 (1993); Towers et al., in Proc. Natl.Acad. Sci. USA 90:6310-6314 (1993); and Zechel et al., in EMBO J.13:1414-1424 (1994)). In contrast to this mode of DNA binding, a growingnumber of receptor-like proteins have been identified which bind as amonomer to a single core-site. The NGFI-b/Nurrl orphan receptors providewell characterized examples of this paradigm (Wilson et al., in Mol.Cell Biol. 13:5794-5804 (1993)).

[0007] Once bound to an HRE, each receptor responds to its signalthrough the C-terminal ligand binding domain (LBD), which binds itscognate hormone with high affinity and specificity (see, for example,Evans, 1988, supra; or Forman and Samuels, 1990, supra). The LBD is acomplex entity containing several embedded subdomains. These include aC-terminal transactivation function (τ2), a series of heptad repeatswhich serve as a dimerization interface and a poorly-delineatedtranscriptional suppression domain (see FIG. 1, and Forman and Samuels,1990, supra).

[0008] The transactivation domain, τ2, consists of approximately 20amino acids with the potential to form an amphipathic α-helix (see Zenkeet al., in Cell 61:1035-1049 (1990); Danielian et al., in EMBO J.11:1025-1033 (1992); Nagpal et al., in EMBO J. 12:2349-2360 (1993); andDurand et al., in EMBO J. 13:5370-5382 (1994)). When linked to aheterologous DNA binding domain, the isolated τ2 domain displaysconstitutive transcriptional activity. However, in the natural contextof the LBD, transcriptional activity requires the addition of ligand.

[0009] The above-described evidence indicates that the LBD functions asa modular unit whose transcriptional activities are controlled byligand. Accordingly, it should be possible for both members of areceptor heterodimer to be simultaneously activated by specific ligandstherefor. However, in spite of this possibility, it has been discoveredthat the ligand-induced transcriptional activities of various receptorsubtypes vary as a function of the partner with which a subtypeparticipates in the formation of a heterodimer. For example, theligand-induced transcriptional activities of RXR are suppressed whencomplexed with RAR and T₃R. This suppression occurs at the level ofligand binding and transcriptional activation. Furthermore, RXRresponsiveness has not been observed with other partners, including VDR.

[0010] Accordingly, the identification of receptor subtypes whichparticipate in the formation of RXR-containing heterodimers, yet retainthe ability to be activated by RXR-selective ligands, would be highlydesirable. The present invention identifies such receptor subtypes andprovides methodology for identifying additional receptor species havingsuch properties.

BRIEF DESCRIPTION OF THE INVENTION

[0011] In accordance with the present invention, it has been discoveredthat RXR can interact productively with Nurrl, a member of the nuclearreceptor superfamily that (in the absence of heterodimerizing partnertherefor) is capable of binding DNA as a monomer (see, for example, Lawet al., in Mol. Endocrinol. 6 :2129-2135 (1992); and Scearce et al., inJ. Biol. Chem. 268:8855-8861 (1993)). As a result of this interaction,the constitutive activity of Nurrl is suppressed, and the resultingcomplex becomes responsive to RXR-selective ligands (e.g., 9-cisretinoic acid). The unique ability of the Nurrl-RXR heterodimer complexto transduce RXR signals establishes a novel response pathway.

[0012] The results described herein suggest that heterodimer formationimparts allosteric changes upon the ligand binding domain (LBD) ofnuclear receptors. These allosteric changes confer transcriptionalactivities onto the heterodimer that are distinct from those of thecomponent monomers. This arrangement permits a limited number ofregulatory proteins to generate a diverse set of transcriptionalresponses to multiple hormonal signals.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 schematically represents the functional domains of nuclearhormone receptors. “DNA” represents the DNA binding domain. “LIGAND”reprsents the large C-terminal ligand binding domain. Dimerization andtransactivation (τ2) functions are embedded within this region, asillustrated.

[0014]FIG. 2 illustrates the differential modulation of RXR response byT₃R (shown in FIG. 2A) and RAR (shown in FIG. 2B).

[0015]FIG. 3 illustrates the differential modulation of RXRtranscriptional activity by the LBDs of T₃R, RAR and VDR.

[0016]FIG. 4A illustrates the differential modulation of RXRtranscriptional activity by the LBD of T₃R, wherein cells treatedaccording to FIG. 3 were additionally treated with T₃ (i. e., T₃Rligand) and LG69 (i. e., an RXR specific ligand) . Normalized reporteractivity was determined and plotted as fold-activation relative tountreated cells.

[0017]FIG. 4B illustrates the differential modulation of RXRtranscriptional activity by the LBD of RAR, wherein cells treatedaccording to FIG. 3 were additionally treated with AM580 (i.e., an RARspecific ligand) and LG69. Normalized reporter activity was determinedand plotted as fold-activation relative to untreated cells.

[0018]FIG. 5 illustrates the ability of T₃R and RAR to suppresstranscription of a constitutively active RXR derivative (i.e.,VP16-RXR).

[0019]FIG. 6 collectively illustrates that the ligand binding acvitivyof RXR is altered by T₃R and RAR.

[0020]FIG. 6A illustrates the binding of LG69 (an RXR specific ligand),at-RA (all-trans retinoic acid, an RAR specific ligand) and Am580 (anRAR specific ligand) to RXR and/or RAR.

[0021]FIG. 6B illustrates that the binding of LG69 to RXR is reduced inRAR-RXR and T₃R-RXR heterodimers.

[0022]FIG. 6C illustrates that competition of [³H] 9-cis RA bound toRXR-RAR heterodimers requires RAR and RXR ligands.

[0023]FIG. 7 collectively demonstrates that a novel Nurrl-RXR complexprovides a signaling pathway for 9-cis retinoic acid.

[0024] Thus, FIG. 7A presents the results of transient transfectionanalysis of GAL-Receptor LBD chimeras in the presence of the RXR LBD.

[0025]FIG. 7B presents transient transfection analysis of full-lengthNurrl and/or RXR.

[0026]FIG. 7C presents a comparison of the responsivity of Nurrl-RXRcomplex, or RXR alone, in the presence and absence of RXR specificligand in the presence of a Nurrl specific response element (NBRE) or anRXR specific response element (CRBPII).

[0027]FIG. 7D demonstrates that the RXR LBD activates through Nurrl butinhibits activation of other receptors.

[0028]FIG. 8 presents an allosteric control model of ligandresponsiveness.

DETAILED DESCRIPTION OF THE INVENTION

[0029] In accordance with the present invention, there is provided aheterodimer complex comprising RXR and a silent partner therefor.

[0030] As employed herein, the term “silent partner” refers to membersof the steroid/thyroid superfamily of receptors which are capable offorming heterodimeric species with RXR, wherein the silent partner ofthe heterodimer is not capable of binding ligand (i.e. , only the RXRco-partner of the heterodimer is capable of binding ligand).

[0031] As employed herein, the phrase “members of the steroid/thyroidsuperfamily of receptors” (also known as “nuclear receptors” or“intracellular receptors”) refers to hormone binding proteins thatoperate as ligand-dependent transcription factors, including identifiedmembers of the steroid/thyroid superfamily of receptors for whichspecific ligands have not yet been identified (referred to hereinafteras “orphan receptors”) . These hormone binding proteins have theintrinsic ability to bind to specific DNA sequences. Following binding,the transcriptional activity of target gene (i.e. , a gene associatedwith the specific DNA sequence) is modulated as a function of the ligandbound to the receptor.

[0032] The DNA-binding domains of all of these nuclear receptors arerelated, consisting of 66-68 amino acid residues, and possessing about20 invariant amino acid residues, including nine cysteines.

[0033] A member of the superfamily can be identified as a protein whichcontains the above-mentioned invariant amino acid residues, which arepart of the DNA-binding domain of such known steroid receptors as thehuman glucocorticoid receptor (amino acids 421-486), the estrogenreceptor (amino acids 185-250), the mineralocorticoid receptor (aminoacids 603-668), the human retinoic acid receptor (amino acids 88-153).The highly conserved amino acids of the DNA-binding domain of members ofthe superfamily are as follows:                           (SEQ ID No 1)Cys - X - X   Cys - X - X - Asp* - X  - Ala* - X -  Gly* - X - Tyr*  -X - X  - X - X -  Cys - X -  X -  Cys -  Lys*  - X - Phe - Phe - X -Arg* - X - X - X  - X - X - X - X - X - X - (X - X -) Cys - X - X - X -X - X - (X - X - X -) Cys - X - X - X - Lys - X - X - Arg - X - X -Cys - X  - X  - Cys  - Arg* - X - X  - Lys* - Cys -  X - X  - X - Gly* - Met;

[0034] wherein X designates non-conserved amino acids within theDNA-binding domain; the amino acid residues denoted with an asterisk areresidues that are almost universally conserved, but for which variationshave been found in some identified hormone receptors; and the residuesenclosed in parenthesis are optional residues (thus, the DNA-bindingdomain is a minimum of 66 amino acids in length, but can contain severaladditional residues).

[0035] Examples of silent partners contemplated for use in the practiceof the present invention are various isoform(s) of Nurrl, HNF4 [see, forexample, Sladek et al., in Genes & Development 4: 2353-2365 (1990)], theCOUP family of receptors [see, for example, Miyajima et al., in NucleicAcids Research 16: 11057-11074 (1988), Wang et al., in Nature 340:163-166 (1989)], COUP-like receptors and COUP homologs, such as thosedescribed by Mlodzik et al., in Cell 60: 211-224 (1990) and Ladias etal., in Science 251: 561-565 (1991), the ultraspiracle receptor [see,for example, Oro et al., in Nature 347: 298-301 (1990)], and the like.

[0036] RXR species contemplated for use in the practice of the presentinvention are selected from RXRα, RXRβ, RXRγ, and the like.

[0037] In accordance with another embodiment of the present invention,there is provided a method to suppress the constitutive activity ofNurrl. Such method comprises contacting Nurrl with at least the ligandbinding domain of RXR.

[0038] In accordance with yet another embodiment of the presentinvention, there is provided a method to render Nurrl-containing cellsinducibly responsive to RXR selective ligands. Such method comprisescontacting such cells with at least the ligand binding domain of RXR.

[0039] In accordance with still another embodiment of the presentinvention, there is provided a method to render RXR-containing cellsresponsive to RXR selective ligands. Such method comprises contactingsaid cells with a silent partner therefor.

[0040] In accordance with a further embodiment of the present invention,there is provided a method for the identification of nuclear receptor(s)which participate as silent partner(s) in the formation of a heterodimerwith RXR. Such method comprises

[0041] introducing into a cell:

[0042] at least the ligand binding domain of a putative silent partnerfor RXR,

[0043] a chimeric construct containing a GAL4 DNA binding domain and atleast the ligand binding domain of RXR, and

[0044] a reporter construct, wherein said reporter construct comprises:

[0045] (a) a promoter that is operable in said cell,

[0046] (b) a GAL4 response element (or a response element for theputative silent partner, when substantially full length putativereceptor is employed), and

[0047] (c) DNA encoding a reporter protein,

[0048] wherein said reporter protein-encoding DNA is operatively linkedto said promoter for transcription of said DNA, and

[0049] wherein said GAL4 response element is operatively linked to saidpromoter for activation thereof, and thereafter

[0050] monitoring expression of reporter upon exposure of theabove-described cell to RXR selective ligand(s).

[0051] In accordance with a still further embodiment of the presentinvention, there is provided a method for identifying ligands selectivefor heterodimers comprising RXR and a silent partner therefor. Suchmethod comprises

[0052] comparing the level of expression of reporter when cellscontaining a reporter construct, RXR and silent partner therefor areexposed to test compound, relative to the level of expression ofreporter when cells containing a reporter construct, RXR and a member ofthe steroid/thyroid superfamily which is not a silent partner thereforare exposed to test compound, and

[0053] selecting those compounds which activate only the combination ofRXR and silent partner therefor.

[0054] The LBD of nuclear hormone receptors is a complex multifunctionalunit containing subdomains for dimerization, transcriptional suppressionand hormone-induced transactivation (Forman and Samuels, 1990, supra).The dimerization domain incudes a series of heptad repeats flanked bysequences required for ligand binding. Thus, the dimerization domain isembedded within the larger LBD. This structural arrangement raises thepossibility that dimerization may serve as an allosteric modulator ofligand binding and transactivation. This possibility has beeninvestigated with the following observations.

[0055] First, dimerization within the LBD is utilized to confertranscriptional suppression upon certain heterodimeric complexes. Thisis exemplified by unliganded T₃R and RAR, which confer transcriptionalsuppression upon RXR. Similarly, in accordance with the presentinvention, it is demonstrated that RXR can suppress constitutiveactivation by Nurrl.

[0056] Second, the intrinsic ligand binding capacity of the LBD can bemodulated by dimerization. This is illustrated by the ability ofunliganded RAR to abrogate the ligand binding activity of RXR. It hasalso been found that T₃R induces a similar suppression, but the presenceof ligand therefor, i.e., T₃, is required for the complete effect. Thus,RXR is seen to serve as a silent partner when participating in the T₃Rand RAR pathways.

[0057] However, not all heterodimeric interactions restrictligand-responsiveness. Indeed, in accordance with the present invention,it is demonstrated that RXR actively confers ligand-responsiveness uponthe Nurrl-RXR heterodimer complex. Similarly, it has previously beenshown that the Drosophila ecdysone receptor (EcR) acquires ligandbinding activity after heterodimerization with USP (Drosophila homologof RXR; see Yao et al., in Nature 366:476-479 (1993)). Thus,differential interactions among receptor LBDs can either restrict,redirect or lead to an acquisition of new ligand binding phenotypes.

[0058] In accordance with the results described herein, a structuralmodel is proposed (see FIG. 8) to account for the observations. In FIG.8, RXR (dark shading) and its partner receptor (e.g., T₃R, RAR or Nurrl(designated “R” in the figure, shown in light shading) initially existas monomers in solution. RXR in monomeric form is capable of bindingligand. RXR-receptor heterodimers then form, driven by the dimerizationinterface that is embedded within the ligand binding domain (LBD) .Subsequent to dimerization, binding of ligand (e.g., 9-cis RA) to RXR ismodestly reduced by T₃R and dramatically reduced by RAR. Addition ofligand for T₃R (e.g., T₃) results in a further reduction in 9-cis RAbinding, while certain retinoids (shown as “RA” in the figure) such asAm580 (an RAR specific ligand) may restore 9-cis RA binding to RXR-RAR.It is of particular note that the Nurrl-RXR heterodimer maintains theability to bind 9-cis RA.

[0059] The above-described structural model relies on the observationthat a major dimerization interface is embedded within the larger LBD.It is proposed that upon dimerization, the structure of the RXR ligandbinding/dimerization domain is altered. Each RXR partner gives rise tounique conformational changes that either maintain or abrogate RXRligand binding activity. Binding of ligand by the partner receptorinduces a conformational change that can be propagated through thedimerization interface onto the LBD of RXR. This model allows one toexplain how the dimerization partner and its specific ligand exertallosteric control over the RXR ligand response.

[0060] In the above-described model, the RXR monomer (or homodimer) iscapable of binding ligand with high affinity. When RXR interacts withone of its non-permissive partners (i.e., T₃R or RAR), its ability tobind ligand is diminished. On the other hand, dimerization of USP/RXRwith EcR promotes high affinity binding of ecdysone to EcR. It isbelieved that these effects are a direct consequence of the localizationof a major dimerization interface within the LBD (see FIGS. 1 and 8).The above-described model predicts that this structural arrangementserves to functionally link dimerization and ligand binding activities.This would then provide a mechanism by which dimerization could exertallosteric control over the ligand response.

[0061] In addition to dimerization, ligand binding by one receptor mayalso result in allosteric modification of its partner. Specifically,binding of ligand to the RXR partner can either restore (as in the caseof RAR) or further decrease (as in the case of T₃R) the ligand bindingpotential of RXR (see FIG. 6). It is already known that upon ligandbinding the cognate receptor undergoes a conformation change (see, forexample, Toney et al., in Biochemistry 32:2-6 (1993)). The resultsprovided herein support the suggestion that ligand-induced conformationchanges in the LBD of one heterodimer partner will be propagated throughthe dimerization interface onto the LBD of the partner. Thus, the modelpresented above can explain how a dimerization partner and its specificligand can exert allosteric control over the RXR ligand response.Similarly, the above-described model can account for the ability ofligand to either promote EcR-USP, (Yao et al., 1993, supra) ordestabilize VDR-RXR and T₃R-T₃R dimers (see, for example, Andersson etal., in Nucleic Acids Res. 20:4803-4810 (1992); Ribiero et al., in Mol.Endocrinol. 6:1142-1152 (1992); Yen et al. , in J. Biol. Chem.267:3565-3568 (1992); MacDonald et al, in Mol. Cell Biol . 13:5907-5917(1993); and Cheskis and Freedman, in Mol. Cell Biol. 14:3329-3338(1994)).

[0062] The restriction of RXR activity within certain heterodimersindicates that 9-cis RA responsiveness is not an obligatory consequenceof heterodimerization with RXR. This allows RXR to function as both areceptor and as a heterodimerization partner, without requiring alltarget genes to be 9-cis RA responsive. This explains the paradox as tohow RXR serves as a common subunit for receptors which displayindependent physiologic effects (e.g. T₃R, RAR, VDR).

[0063] In contrast, the ability of RXR to transduce signals whencomplexed with Nurri suggests an alternative pathway for 9-cis RAsignaling. Nurrl expression is induced by physiological stimuli (seeDavis and Lau, in Mol. Cell Biol. 14:3469-3483 (1994)) includingmembrane depolarization and liver regeneration (Scearce et al., 1993,supra). Based on the results presented herein, it is clear that RXRcontributes to the regulation of these events.

[0064] Unlike previously described heterodimers, RXR functionallyinteracts with Nurrl in the absence of RXR-specific DNA contacts (seeFIG. 7D) . Indeed, the ability to tether to a DNA bound monomer is adistinguishing feature of the Nurrl-RXR heterodimer complex. As aresult, an RXR mutant that is deficient in DNA binding activates throughNurrl while it inhibits other receptor heterodimers (see FIG. 7D).

[0065] In accordance with the present invention, there are providedmethods for the modulation of Nurrl expression induced by physiologicalstimulus of a subject. Such method comprises administering to thesubject an effective amount of a composition comprising at least theligand binding domain of RXR. Physiological stimuli contemplated fortreatment in accordance with the present invention include any eventwhich induces production of calcium ions, cyclic AMP, ACTH, and thelike.

[0066] The invention will now be described in greater detail byreference to the following non-limiting examples.

Example 1 Cell Culture and Transfection

[0067] CV-1 cells were grown in Dulbecco's Modified Eagle's mediumsupplemented with 10% resin-charcoal stripped (Samuels et al.,Endocrinology 105:80-85 (1979)) fetal bovine serum, 50 U/ml penicillin Gand 50 μg/ml streptomycin sulfate (DMEM-FBS) at 37° C. in 5% CO₂. Oneday prior to transfection, cells were plated to 50-80% confluence usingphenol-red free DMEM-FBS. Cells were transfected by lipofection usingN-{2-(2,3)-dioleoyloxy)propyl-N,N,N-trimethyl ammonium methyl sulfate}according to the manufacturer's instructions (DOTAP, BoehringerMannheim). After 2 hours, the liposomes were removed and cells treatedfor 40 hours with phenol-red free DMEM-FBS alone or with the followingligands: 100-300 nM T₃ (L-triiodothyronine), 100 nM LG69 (4-{1- (3, 5,5, 8, 8-pentamethyl-5, 6, 7,8-tetrahydro-2-napthalenyl)-1-propenyl}benzoic acid), 50-100 nM Am580(4-(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-napthamido) benzoicacid) or 100 nM VD₃ (1α, 25-dihydroxyvitamin D₃) . Cells were harvestedand assayed for luciferase and β-galactosidase activity. All points wereperformed in triplicate in each experiment and varied by less than 10%.Each experiment was repeated three or more times with similar results.

Example 2 Expression and Reporter Constructs

[0068] For luciferase assays, response elements with HindIII overhangswere cloned into the HindIII site of the TK-LUC reporter which containsthe Herpes virus thymidine kinase promoter (−105/+51). Response elementswith the underlined consensus hexanucleotide sequence were as follows:UAS_(G) × 4 (i.e., 4 copies of the following sequence):                                       SEQ ID NO:2 5′-CGACGGAGTACTGTCCTCCGAGCT; IRO = TREp (i.e., 1 & 2 copies of the followingsequence):                                        SEQ ID NO:35′-TCAGGTCA TGACCTGAG; DR4 × 2                                       SEQ ID NO:45′-A A A G G T C A C G A A A G G T C A CCATCCCGGGA AAAGGTCACGAAAGGTCACC;DR5                                        SEQ ID NO:55′-CAGGTCA-CCAGGAGGTCAGAG; DR5 × 2                                       SEQ ID NO:65′-A A A G G T C A C C G A A A G G T C A CCATCCCGGGAAAAGGTCACCGAAAGGTCACC; ER8                                        SEQID NO:7 5′-TGACCTTTCTCTCC AGGTCA; NERE × 3 (i.e., 3 copies of thefollowing sequence):                                        SEQ ID NO:85′-GAGTTTAAAAGGTCA TGCTCAATTTTC; CRBPIT                                       SEQ ID NO:95′-GTCACAGGTCACAGGTCACAGGTCACAGTTCA; MLV-DR4 × 2 (i.e., 2 copies of thefollowing sequence):                                       SEQ ID NO:105′-AAGGTTCACGAGGTTCACGT.

[0069] All mammalian expression vectors were derived from pCMX (Umesonoet al., 1991, supra) which contains the CMV promoter/enhancer followedby a bacteriophage T7 promoter for transcription in vitro. pCMXexpression vectors for T₃R₆₂ , hRARα (Umesono et al., 1991, supra) andhRXRα (Yao et al., 1993, supra) were used as previously described.CMX-Nurrl (provided by Thomas Perlmann), an expression vector forfull-length mouse Nurrl, was cloned by inserting the BglII-XhoI fragmentfrom pBS34-1 (excised from λZAP34) (see Law et al., 1992, suPra) intopCMX. The VP16-RXR fusion contains the 78 amino acid transactivationdomain of Herpes VP16 from pVP16Cl (Novagen) fused N-terminal to thefull-length hRXRα.

[0070] GAL4 fusions were made by fusing the following receptor ligandbinding domains to the C-terminal end of the yeast GAL4 DNA bindingdomain (amino acids 1-147) from pSG424 (see Sadowski and Ptashne, inNucleic Acids Res. 17:7539 (1989)): human RXRα LBD (Glu 203 - Thr 462);mouse Nurrl (Cys 318 - Phe 598); human T₃Rβ (Leu 173 - Asp 456); humanRARα (Glu 156 - Pro 462); and human VDR (Glu 92 - Ser 427). The LBDexpression constructs contain the SV40 TAg nuclear localization signal(APKKKRKVG; SEQ ID NO:11) fused upstream of the human T₃Rβ LBD (Leu173 - Asp 456), HRARα LBD (Glu 156 - PRO 462) or the human RXRα LBD (Glu203 -Thr 462). CMX-βgal contains the E. coli β-galactosidase codingsequences derived from pCH110 (Pharmacia) cloned into PCMX.

[0071] In the left panel of FIG. 5, CV-1 cells were transfected with thefollowing plasmids: IRO TK-LUC (300 ng/10⁵ cells), CMX-βgal (500 ng/10⁵cells) alone (−) or with CMX-VP16-RXRα (100 ng/10⁵ cells) and/orCMX-hRARα (50 ng/10⁵ cells) as indicated. No ligand treatment wasemployed. Luciferase activity was normalized to the β-galactosidaseinternal control. In each experiment, the normalized activity obtainedin the presence of VP-RXR, T₃R or RAR is plotted as activity relative tothe reporter alone, which was defined to have a relative activity of 1.

Example 3 Ligand Binding Assays

[0072] Bacterially expressed proteins were used for ligand bindingassays. GST-hRXRα (see Mangelsdorf et al., in Cell 66: 555-561 (1991)),chicken T₃Rα1 (see Forman et al., in Mol. Endocrinol. 6:429-442 (1992))and human RARα (Forman et al., 1992, supra) were expressed and purifiedto near homogeneity as previously described. GST-RXR (150 ng) or a GSTcontrol (150 ng) were incubated with or without approximately 500 ng ofT₃R or RAR in the presence of 50 nM [³H]-ligands (LG69, 56 Ci/mmol;at-RA, 49 Ci/mmol; 9-cis RA, 29 Ci/mmol) , 3 ng/μl poly dI-dC, 50fmol/μl of the indicated oligonucleotide, 10 μl of 50% (v/v)epoxy-linked glutathione-sepharose (Sigma) in ligand binding buffer (25mM Tris, pH 7.8, 0.5% CHAPS, 100 mM KCl, 8% Glycerol, 1 mM DTT).

[0073] Where indicated (see, for example, FIG. 6) unlabeled ligands wereadded as follows: LG69, 2 μM; Am580, 2 μM; T₃, 1 μM. The reaction wasmixed for 30 minutes at 25° C. and then chilled to 4° C. for 10 minutes.The glutathione-sepharose beads were washed three times in ligandbinding buffer and the amount of [³H] bound was determined in a liquidscintillation counter. Background binding was determined with the GSTcontrol and represented 3-5% of the total binding seen with GST-RXR.

Example 4 RXR Responsiveness is Diminished in T₃R-RXR and RAR-RXRHeterodimers

[0074] Since T₃R and RAR function as heterodimers with RXR, RXRresponsiveness was examined in the context of RXR-T₃R and RXR-RARheterodimers. Attention is directed to FIG. 2, wherein transienttransfection analysis of T³R-RXR and RAR-RXR heterodimers is described.Reporter constructs employed contain the HRE indicated in the figure,cloned upstream of the TK-LUC reporter. In the left panel of the figure,CV-1 cells were transfected with the following plasmids: HRE×2 - TK-LUC(300 ng/10⁵ cells), CMX-hT₃Rβ (20 ng/10⁵ cells), CMX-hRXRα (20 ng/10⁵cells) and the internal control CMX-βgal (500 ng/10⁵ cells) . Cells weretreated without ligand or with 100 nM T₃, 100 nM LG69 or 100 nM T₃+100nM LG69.

[0075] In the right panel of FIG. 2, cells were transfected with HRE×1TK-LUC (300 ng/10⁵ cells), CMX-hRARα (50 ng/10⁵ cells) CMX-hRXRα (50ng/10⁵ cells) and CMX-βgal (500 ng/10⁵ cells) . Cells were treatedwithout ligand or with 50 nM Am580, 100 nM LG69 or 50 nM Am580+100 nMLG69. Normalized luciferase activity was determined and plotted asfold-activation relative to untreated cells.

[0076] Although cells transfected with both T₃Rβ and RXRα expressionvectors were responsive to T₃, they were surprisingly not responsive tothe RXR specific ligand LG69 (see FIG. 2; Boehm et al., in J. Med. Chem.37:408-414 (1994) ) . Treatment of these cells with both T₃ and LG69 didnot result in further stimulation of the T₃ response, rather theresponse to T₃ was somewhat reduced. Similarly, cells simultaneouslytransfected with RARα and RXRα expression vectors responded to theRAR-specific ligand Am580, but remained unresponsive to LG69. Incontrast, treatment with Am580+LG69 resulted in increasedtranscriptional activity over that seen with AM580 alone.

Example 5 Suppression of RXR Activity is Mediated by the LBD

[0077] Since RXR homodimers are activated RXR agonists, the resultspresented above suggest that RXR activity is suppressed in unligandedRXR-T₃R and RXR-RAR heterodimers. It is suspected thatheterodimerization within the LBD (see FIG. 1) could induce anallosteric change in the RXR LBD that blocks its ability to bind ligandand/or transactivate. To test this hypothesis, a system was developed toexamine the responsiveness of RXR-containing heterodimers in a mannerthat relies solely on interactions between the LBDs.

[0078] Thus, a chimeric protein was constructed containing the yeastGAL4 DBD linked to the RXR LBD (GAL-RXR). The ability of thisRXR-chimera to respond to LG69 was initially examined in the presence oftruncated receptors containing the LBDs of T₃R or RAR. Thus, transienttransfection analysis of GAL-RXR LBD was carried out in the presence ofT₃R, RAR or VDR LBDs. Reporter constructs contained 4 copies of theUAS_(G) cloned upstream of the TK-LUC reporter. CV-1 cells weretranfected with UAS_(G) ×4 TK-LUC (300 ng/10⁵ cells), CMX-GAL-RXR (100ng/10⁵ cells), CMX-βgal (500 ng/10⁵ cells) alone or with either CMX-T₃RLBD, CMX-RAR LBD or CMX-VDR LBD (100 ng/10⁵ cells). Followingtransfection, cells were treated without ligand or with 100 nM LG69, 100nM T_(3,) 50 nM Am580 or 100 nM VD₃. Normalized luciferase activity wasdetermined and plotted as reporter activity (see FIG. 3).

[0079] Although GAL-RXR activated the UAS_(G) reporter in response toLG69, the absolute levels of induced and uninduced activity weredramatically suppressed by both T₃R and RAR LBDs (see FIG. 3) Incontrast, the VDR LBD failed to suppress RXR responsiveness. Theseresults indicate that suppression of RXR by unliganded T₃R and RAR ismediated solely by interactions between the LBDs of these receptors.

[0080] These results are consistent with previous experiments which haveshown that receptor LBDs remain tethered to the GAL-RXR LBD in cells(see, for example, Nagpal et al., 1993, supra). Thus, it was next soughtto determine whether the tethered LBDs can activate transcription inresponse to their specific ligands. As seen in FIG. 3, the T₃R, RAR andVDR LBDs conferred ligand-dependent activation upon GAL-RXR, but notGAL4 alone. Thus, receptor LBDs tethered to RXR provide all thefunctions required for ligand-dependent transcriptional activation inthe absence of direct DNA contact.

[0081] The experiment described with respect to FIG. 3 was alsoperformed with the combination of RXR-specific ligand (e.g., LG69) andT₃R or RAR specific ligand (see FIG. 4, which illustrates thedifferential modulation of RXR transcriptional activity by the LBD ofT₃R. Thus, cells treated according to the procedure described above withrespect to FIG. 3 were additionally treated with 100 nM T₃+100 nM LG69(see FIG. 4A) or 50 nM AM580+100 nM LG69 (see FIG. 4B). Normalizedluciferase activity was determined and plotted as fold-activationrelative to untreated cells.

[0082] In order to compare the effects of T₃R and RAR LBDs on LG69inducibility of GAL-RXR, these data were re-plotted as fold-induction.Comparison of FIGS. 2 and 4 indicate that the effects of ligand-occupiedT₃R and RAR are qualitatively similar, regardless of whether thefull-length receptors or their LBDs are used. Note that the T₃R LBD ledto a coordinate reduction in both basal and LG69-induced activities ofGAL-RXR, hence the fold response to LG69 was only modestly inhibitedfrom 69-fold (see FIG. 4A, GAL-RXR alone) to 57-fold by the T₃R LBD(FIG. 4B, GAL-RXR+T₃R LBD) . Addition of T₃ resulted in strongactivation of T₃R and the combination of T₃+LG69 resulted in slightlyless activity than with T₃ alone. In contrast to T₃R, unliganded RAR LBDstrongly suppressed the fold-responsiveness of GAL-RXR to LG69.Treatment with Am580+LG69 resulted in increased transcriptional activityover that seen with AM580 alone suggesting that RXR responsiveness toLG69 may be restored by addition of the PAR agonist Am580 (FIG. 4B).

Example 6 RAR and T₃R Differentially Suppress the Ligand BindingActivity of RXR

[0083] In addition to decreasing basal and activated transcription, RARalso blocks the ability of RXR to respond to its ligand. Thus, thepossibility that RXR is incapable of binding ligand when tethered to RARwas examined. A bacterially expressed glutathione-S-transferase-RXRafusion protein (GST-RXR) was incubated with recombinant T₃R or RAR inthe presence of radiolabeled RXR ligands. The amount of ligand bound toRXR or RXR-containing heterodimers was quantitated usingglutathione-sepharose as an affinity probe. In the left panel of FIG.6A, purifed GST-hRXRα was incubated with 50 nM [³H] LG69 (56 Ci/mmol)and the optimized PAR reponse element 5′-GCAAA AGGTCA AAAAG AGGTCATGC-3′; SEQ ID NO:12; Kurokawa et al., Genes Dev. 7:1423-1435 (1993))alone or with 2 μM LG69, 2μM Am580. In the right panel of FIG. 6A,purified GST-hRXRα and the RAR response element were incubated with 25nM [³H] at-RA (49 Ci/mmol) without or with 500 ng of hRARα. The amountof specifically bound [³H] label was then determined employing standardtechniques as previously described.

[0084] As expected, binding of [³H] LG69 to GST-RXR was specificallycompleted by unlabeled LG69, but not by the RAR-specific ligand Am580(see FIG. 6A, right panel); specific binding of [³H] all-trans RA(at-RA) was observed when GST-RXR was mixed with excess PAR (see FIG.6A, right panel) . A quantitation of the amount of specifically bound[³H] LG69, [³H] at-RA or (¹²⁵I] T₃ indicates that GST-RXR could besaturated with approximately equimolar amounts of RAR or T₃R,respectively. Electrophoretic mobility shift experiments indicate thatligands do not alter the binding activity of T₃R-RXR or RAR-RXRheterodimers.

[0085] Next, the ligand binding activity of RXR was examined in thepresence of RAR-T₃R. Thus, purified GST-hRXRα and 50 nM [³H] LG69 (56Ci/mmol) were incubated alone or with 500 ng of hRARα or chicken T₃Rαland the optimized RAR response element or the optimized T₃R responseelement 5′-GCAAA AGGTCA AATA AGGTCA CGT-3′; SEQ ID NO:13; Kurokawa etal., supra), respectively. Where indicated, unlabeled T₃ was added to aconcentration of 1 μM. Specifically bound [³H] LG69 was determined.

[0086] Surprisingly, addition of RAR resulted in a dramatic (<85%)decrease in the amount of [³H] LG69 bound to GST-RXR (see FIG. 6B),indicating that the ligand binding potential of RXR is reduced in theRXR-RAR heterodimer. These findings account for the ability ofunoccupied RAR to suppress the ligand inducibility of RXR (see FIG. 4B).

[0087] Similar experiments were performed on the RXR-T₃R heterodimer. Incontrast to PAR, unliganded T₃R led to a modest reduction in [³H] LG69binding. However, ligand binding was strongly diminished upon additionof T₃ (FIG. 6B) . These findings are consistent with the observationthat unoccupied T₃R results in a modest suppression of RXR inducibility,whereas no induction is elicited when T₃R is occupied by T₃ (FIG. 4B)

[0088] The transfection experiments summarized in FIGS. 2 and 4Bindicate that RAR-RXR heterodimers exhibit RXR responsiveness only inthe presence of an RAR ligand, suggesting that RXR binding activity maybe restored by RAR ligands. To test this hypothesis, the observationthat 9-cis RA binds with high affinity to both RAR and RXR (Allegrettoet al., 1993; Allenby et al., 1993) was applied as follows. Thus,GST-RXR/RAR heterodimers were allowed to form in the presence of [³H]9-cis RA. Reactions were performed as described above with reference toFIG. 6A, using both GST-hRXRα and hRARα with 50 nM [³H] 9-cis RA (29Ci/mmol) . Specifically bound [³H] 9-cis RA was determined in theabsence or presence of 2 μM LG69 and/or 2 μM Am580. In all experiments,maximal binding was in the range of 200-300 fmol of [³H] ligand.

[0089] Although Am580 fully competed with [³H] at-RA for binding toGST-RXR/RAR heterodimers (FIG. 6A, right panel), Am580 resulted in onlya partial decrease in [³H] 9-cis RA binding (see FIG. 6C) . Nearlycomplete competition was observed by addition of both Am580 and theRXR-specific ligand LG69 (see FIG. 6C), suggesting that RXR can bindligand, provided the RAR LBD is occupied. These findings are consistentwith the restoration of RXR responsiveness in RAR-occupied heterodimers(FIG. 4B).

Example 7 Identification of a Novel RXR-permissive Heterodimer

[0090] Since RXR serves as a silent partner in the T₃R and RAR pathways,it was next investigated whether RXR could serve as an active componentin other complexes. To search for such complexes, the LBD of a number ofnuclear receptors were fused to the GAL4 DBD, and tested to determinewhether the RXR LBD could confer LG69 responsiveness upon these GAL-LBDchimeras. Thus, CV-1 cells were transfected with UAS_(g)×4 TK-LUC (300ng/10⁵ cells), CMX-βgal (500 ng/10⁵ cells) and the indicatedCMX-GAL-receptor LBD construct (100 ng/10⁵ cells) with or withoutCMX-RXR LBD (100 ng/10⁵ cells). Following transfection, cells weretreated without ligand or with 100 nM LG69. Normalized luciferaseactivity was determined and plotted as fold-activation relative tountreated cells.

[0091] As expected, LG69 responsiveness was not seen when the RXR LBDwas expressed alone, or with GAL-T₃R and GAL-RAR (see FIG. 7A).Similarly, LG69 inducibility was not observed with chimeras containingthe LBDs of VDR (see FIG. 7A) or several other members of the nuclearreceptor superfamily. Unexpectedly, strong responsiveness to LB69 wasobserved when the RXR-LBD was co-expressed with a GAL-Nurrl chimera (seeFIG. 7A). These results suggest that the LBDs of Nurrl and RXR form anovel heterodimer complex which promotes potent RXR responsiveness.

[0092] Nurrl (also known as RNR-1, NOT, HZF-3), the β isoform of NGFI-b(also known as nur77, N10, NAK-1, TR3), is reported to be aconstitutively active orphan receptor that binds as a high-affinitymonomer to an AA-AGGTCA core-site (NBRE) (see, for example, Law et al.,1992, supra; Wilson et al., 1992, supra; Scearce et al., 1993, supra;and Wilson et al., 1993, supra). This prompted further investigation asto whether full-length Nurrl and RXR could interact productively on theNBRE.

[0093] Thus, CV-1 cells were tranfected with NBRE×3 TK-LUC (300 ng/10⁵cells) , CMX-βgal (500 ng/10⁵ cells), alone or with CMX-Nurrl (100ng/10⁵ cells) and CMX-hRXRα (100 ng/10⁵ cells) as indicated in FIG. 7B.Following transfection, cells were treated with or without 100 nM LG69.Normalized luciferase activity was determined and plotted as reporteractivity.

[0094] Consistent with published results (see, for example, Scearce etal., 1993, supra), Nurrl constitutively activates the NBRE reporter (seeFIG. 7B), but was not responsive to LG69 (FIG. 7B). RXR, which does notbind to the NBRE, did not activate this reporter. However, when Nurrland RXR are co-expressed, the constitutive activity of Nurri issuppressed, and the complex becomes strongly responsive to LG69 (FIG.7B) . Similar results were obtained with RXRα, RXRβ and RXRγ.

[0095] The ability of the Nurrl-RXR heterodimer complex to transduce RXRsignals suggested the desirability of comparing the activity of thiscomplex with that of RXR on an established RXR response element (CRBPII,cellular retinol binding protein II; see Mangelsdorf et al., 1991,supra). Using sub-optimal amounts of RXR-expression vector, the CRBPIIreporter was compared with a 3-copy NBRE reporter as follows. Cells weretransfected as described with respect to FIG. 7B, but with a 5-foldlower amount of CMX-hRXRα (20 ng/10⁵ cells). CRBPII TK-LUC (300 ng/10⁵cells) was used where indicated.

[0096] Since RXR was limiting in this assay, only minimal activation ofthe CRBPII reporter was observed (see FIG. 7C) . In contrast, Nurrl-RXRdisplayed a potent response to LG69, despite the fact that the NBREreporter contains 1 less core-binding site than CRBPII (see FIG. 7C).Thus, Nurrl-RXR can efficiently transduce RXR signals. However, unlikeother heterodimers, the Nurrl-RXR complex is strongly responsive to LG69and 9-cis RA, suggesting that this complex establishes a novel signalingpathway for 9-cis RA.

Example 8 Nurrl Does not Require the RXR DBD for Coupling

[0097] The Nurrl-RXR complex is unique in several ways. First, the NurrlDBD recognizes its response element in the absence of RXR (see, forexample, Wilson et al., 1992, supra; Scearce et al., 1993, supra; andWilson et al., 1993, supra) . Second, the monovalent NBRE serves as aresponse element for a multimeric Nurrl-RXR complex (see FIG. 7B). Theseobservations raise the possibility that RXR associates with NBRE-boundNurrl in the absence of RXR-specific DNA contacts. Such behavior wouldbe in sharp contrast with T₃R, RAR and VDR, which rely on RXR-specificcontacts to recognize hormone response elements. Indeed, RXR mutantslacking the DBD associate with wild-type RAR; however, these complexesdo not bind DNA or activate transcription (see Minucci et al., in Mol.Cell Biol. 14:360-372 (1994)).

[0098] This prompted an investigation of the question of whether the RXRDBD is required for activation through the Nurrl pathway. Thus, CV-1cells were transfected with TK-LUC reporters (300 ng/10⁵ cells),CMX-βgal (500 ng/10⁵ cells) and the indicated CMX-receptor construct (20ng/10⁵ cells; see FIG. 7D) with or without CMX-RXR-LBD (100 ng/10⁵cells) . The following receptor, reporter, ligand combinations wereused: Nurrl, NBRE×3, 100 nM LG69; hT₃Rβ), MLV×2, 100 nM T₃; hRARα,DR5×2, 100 nM Am580; hVDR, SPP1×3, 100 nM VD₃. Normalized luciferaseactivity was determined and plotted as percent of maximalfold-activation where 100% is defined as the fold activation by T₃R,RAR, VDR, the RXR LBD, or Nurrl+RXR LBD. The actual fold-activationvalues are shown above each bar in the figure.

[0099] As shown in FIG. 7D, the RXR LBD is sufficient to confer strongLG69 responsiveness upon Nurrl. In contrast, the RXR LBD acts as adominant-negative inhibitor of wild-type VDR, T₃R and RAR (FIG. 7D).These findings indicate that the RXR DBD is not required forligand-dependent activation of Nurrl-RXR, a property that furtherdistinguishes this novel complex from previously describedRXR-containing complexes.

[0100] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1 14 71 amino acids amino acid single linear 1 Cys Xaa Xaa Cys Xaa XaaAsp Xaa Ala Xaa Gly Xaa Tyr Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa CysLys Xaa Phe Phe Xaa Arg Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa XaaXaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45 Xaa Xaa Xaa Lys XaaXaa Arg Xaa Xaa Cys Xaa Xaa Cys Arg Xaa Xaa 50 55 60 Lys Cys Xaa Xaa XaaGly Met 65 70 24 base pairs nucleic acid single linear 2 CGACGGAGTACTGTCCTCCG AGCT 24 17 base pairs nucleic acid single linear 3 TCAGGTCATGACCTGAG 17 48 base pairs nucleic acid single linear 4 AAAGTCACGAAAGGTCACCA TCCCGGGAAA AGGTCACGAA AGGTCACC 48 21 base pairs nucleic acidsingle linear 5 CAGGTCACCA GGAGGTCAGA G 21 51 base pairs nucleic acidsingle linear 6 AAAGGTCACC GAAAGGTCAC CATCCCGGGA AAAGGTCACC GAAAGGTCAC C51 20 base pairs nucleic acid single linear 7 TGACCTTTCT CTCCAGGTCA 2028 base pairs nucleic acid single linear 8 GAGTTTAAAA GGTCATGCCTCAATTTTC 28 32 base pairs nucleic acid single linear 9 GTCACAGGTCACAGGTCACA GGTCACAGTT CA 32 20 base pairs nucleic acid single linear 10AAGGTTCACG AGGTTCACGT 20 9 amino acids amino acid single linear 11 AlaPro Lys Lys Lys Arg Lys Val Gly 1 5 25 base pairs nucleic acid singlelinear 12 GCAAAAGGTC AAAAAGAGGT CATGC 25 24 base pairs nucleic acidsingle linear 13 GCAAAAGGTC AAATAAGGTC ACGT 24 8 base pairs nucleic acidsingle linear 14 AAAGGTCA 8

That which is claimed is:
 1. A method to suppress the constitutiveactivity of Nurrl, said method comprising contacting Nurrl with at leastthe ligand binding domain of RXR.
 2. A method according to claim 1wherein the ligand binding domain of RXR is selected from RXRα, RXRβ orRXRγ.
 3. A method to render NurrI-containing cells inducibly responsiveto RXR selective ligands, said method comprising contacting said cellswith at least the ligand binding domain of RXR.
 4. A method according toclaim 3 wherein the ligand binding domain of RXR is selected from RXRα,RXRβ or RXRγ.
 5. A method to render RXR-containing cells responsive toRXR selective ligands, said method comprising contacting said cells witha silent partner therefor.
 6. A method according to claim 5, whereinsaid silent partner is an isoform of Nurrl.
 7. A method for theidentification of nuclear receptor(s) which participate as silentpartner(s) in the formation of a heterodimer with RXR, said methodcomprising introducing into a cell: at least the ligand binding domainof a putative silent partner for RXR, a chimeric construct containing aGAL4 DNA binding domain and at least the ligand binding domain of RXR,and a reporter construct, wherein said reporter construct comprises: (a)a promoter that is operable in said cell, (b) a GAL4 response element,and (c) DNA encoding a reporter protein, wherein said reporterprotein-encoding DNA is operatively linked to said promoter fortranscription of said DNA, and wherein said GAL4 response element isoperatively linked to said promoter for activation thereof, andthereafter monitoring expression of reporter upon exposure of theabove-described cell to RXR selective ligand(s).
 8. A method for theidentification of nuclear receptor(s) which participate as silentpartner(s) in the formation of heterodimer(s) with RXR, said methodcomprising introducing into a cell: a putative silent partner for RXR,at least the ligand binding domain of RXR, and a reporter construct,wherein said reporter construct comprises: (a) a promoter that isoperable in said cell, (b) a response element for said putative silentpartner, and (c) DNA encoding a reporter protein, wherein said reporterprotein-encoding DNA is operatively linked to said promoter fortranscription of said DNA, and wherein said response element for saidputative silent partner is operatively linked to said promoter foractivation thereof, and thereafter monitoring expression of reporterupon exposure of the above-described cell to RXR selective ligand(s). 9.A method according to claim 8 wherein the response element for theputative silent partner has the sequence AAAGGTCA.
 10. A method foridentifying ligands selective for heterodimers comprising RXR and asilent partner therefor, said method comprising comparing the level ofexpression of reporter when cells containing a reporter construct, RXRand silent partner therefor are exposed to test compound, relative tothe level of expression of reporter when cells containing a reporterconstruct, RXR and a member of the steroid/thyroid superfamily which isnot a silent partner therefor are exposed to test compound, andselecting those compounds which activate only the combination of RXR andsilent partner therefor.